Ifn γ

Cell culture media were purchased from Gibco (Carlsbad, USA), and their supplements provided by PAA Laboratories GmbH (Cölbe, Germany). Prokaryotic medium and supplements were ordered from Clontech (Takara, Japan) and Roth GmbH (Karlsruhe, Germany). Auranofin (AF), LPS, tetradecanoylphorbol acetate (TPA), hydrogen peroxide (H₂O₂), dimethylsulfoxide (DMSO), and the PPARγ ligands rosiglitazone and 2-chloro-5-nitrobenzanilide (GW9662) were purchased from Sigma-Aldrich (St. Louis, USA). IFNγ was ordered from BioVision Inc. (Milpitas, USA) and IL4 from Peprotech (Hamburg, Germany). Enzymes were obtained from New England Biolabs (Ipswich, UK) and polymerases from Agilent Technologies Deutschland GmbH (Böblingen, Germany) and Clontech (Takara, Japan). Further chemicals, if not indicated otherwise, were ordered from AppliChem GmbH (Darmstadt, Germany), Merck KGaA (Darmstadt, Germany), Promega GmbH (Mannheim, Germany), Roche Diagnostics (Basel, Switzerland), and Sigma-Aldrich.

Fetal bovine serum and Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin, streptomycin used in the cell culture, and ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Gibco, Thermo Fisher Scientific, Inc. (Billings, MT, USA). IFN-γ was purchased from BioVision, Inc. (Milpitas, CA, USA) and TNF-α antibody was purchased from Signalway Antibody LLC (Greenbelt, MD, USA). Paraformaldehyde, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and DNCB were obtained from Sigma-Aldrich Chemical Corporation (St. Louis, MO, USA). MTT, sodium dodecyl sulfate (SDS), and Triton X-100 were purchased from USB Corporation (Douglasville, AG, USA). Acetone and isopropanol were purchased from J.T. Baker Co. (Phillipsburg, NY, USA). Western Bright ECL kit was purchased from Advansta Inc. (Menlo Park, CA, USA). The chemicals used in the experiments were of reagent grade. Antibodies including human IL-1β (EH0185), human IL-6 (EH0201), mouse TNF-α (EM0183), and mouse TSLP (EM0201) ELISA kits were purchased from Wuhan Fine Biotech Co., Ltd. (Wuhan, China).

Cells were seeded in 96 well tissue culture plates (10⁴/well) for cytotoxicity and total nitrite determination assays, 12 well tissue culture plates (10⁵/well) for real-time PCR measurements, 6 well tissue culture plates (10⁶/well) for Western blot analysis, and onto glass coverslips placed into 24 well tissue culture plates (2 × 10⁴/well) for immunofluorescence. The next day, cells were pretreated with either plant extracts or test compounds, including vanillic acid, α-amyrin, β-caryophyllene, trans-ferulic acid, scopoletin (Sigma-Aldrich, Budapest, Hungary), and spilanthol (Santa Cruz) at the indicated concentrations for 2 h followed by treatment with LPS (10 ng/mL) (Sigma–Aldrich, Budapest, Hungary) and IFNγ (10 ng/mL) (Bio-Vision) for 24 h. Test compounds were first dissolved in DMSO (stock solution) and were then further diluted in culture medium for cell-based experiments. DMSO concentrations were at or below 1% (v/v) in cell cultures which did not have any effect on the parameters measured.

Epigallocatechin gallat (EGCG), Stattic and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, USA). IFNγ was from BioVision (Milpitas, USA). IL-4 was from Peprotech (Hamburg, Germany). IL-27 was obtained from Biolegend (Koblenz, Germany), IL-27 neutralizing antibody was from Invitrogen (Carlsbad, USA), and the IgG2a istotype control was from BioXCell (West Lebanon, USA). Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were from ImmunoTools (Friesoythe, Germany). All reagents were dissolved according to the manufacturer's instructions.

Prostatic sections were fixed overnight in a 10% buffered paraformaldehyde solution. The tissues were ethanol-dehydrated and embedded in paraffin wax. Sections of 5 µm were cut with microtome, deparaffinized at 70°C for 20 min, incubated 5 minutes each in xylene, ethanol-xylene, and 100% ethanol. Finally, the samples were washed with distilled water to be processed for immunohistochemical analysis. Immunolabeling was done by using Mouse/Rabbit PolyDetector Plus HRP/DAB (BSB 0259; BioSB) Kit according to the manufacturer's instructions. The antibodies used were TNF-α (50 µg/mL) (Abcam 1793; Massachusetts, USA), IFN-γ (50 µg/mL) (Abcam 7740; Massachusetts, USA), CD45 (1 µg/mL) (Abcam 10558, Massachusetts, USA), IL-17 (1 µg/mL) (Abcam 79056, Massachusetts, USA), IL-6 (74 µg/mL) (Abcam 6672; Massachusetts, USA), IL-4 (0.5 µg/Ml) (Abcam 11524; Massachusetts, USA), and PCNA (100 µg/mL) (Santa Cruz-53407, Texas, USA). After the immunohistochemistry procedure, microphotographs were taken and the relative expression percentages were semiquantified in the Image-Pro Premier software 9.1.